The enteric nervous system (ENS) is the division of the autonomic nervous system that resides within the gut wall. The ENS controls gastrointestinal (GI) motility, secretion and local blood flow. The ENS can perform these complex functions because it contains all the neuronal elements (sensory neurons, interneurons and motorneurons) required for GI reflexes and integration. The ENS contains 14 different types of neurons that release many different neurotransmitters. There are also multiple receptor subtypes for each neurotransmitter. In addition, synapses in the ENS may be coded by the neurotransmitters released from presynaptic nerve terminals and by receptors expressed by postsynaptic cells. The proposed studies will use intracellular electrophysiological, immunohistochemical and molecular biological methods to study enteric synaptic transmission. There are 3 specific aims in this proposal. Specific aim 1 will focus on inhibitory neurotransmission to the muscle layers. Inhibitory motorneurons release ATP and nitric oxide which relax GI smooth muscle. These studies will test the hypothesis that release of ATP and NO from the same nerve terminal are under the control of different Ca2+ channel types. Specific aim 2 will focus on Ca2+ channels expressed by interneurons in the myenteric plexus. Interneurons which project in an oral-anal direction release acetylcholine (ACh) and ATP as fast synaptic transmitters, while neurons that project in an anal-oral direction release ACh. These studies will test the hypothesis that R-, N- and P/Q type Ca2+ channels are expressed by neurons in the orally-projecting pathway while only N- and P/Q type Ca2+ channels are expressed by nerve terminals in the anally-projecting pathway. Specific aim 3 will focus on postsynaptic interactions between nicotinic acetylcholine receptors (nAChRs), P2X receptors for ATP, a K+ channel and muscarinic cholinergic and P2Y purinergic receptors. This aim will test the hypothesis that these receptors and ion channels are clustered in a signaling complex in myenteric neurons and this close clustering permits very precise and timely modulation of synaptic excitation. Significance: Disturbances in enteric synaptic mechanisms contribute to GI motility disorders. Changes in the function of enteric neurons and their synapses might also contribute to visceral pain. Therefore, a more complete understanding of enteric neural circuits and synaptic transmission would provide insights into the pathophysiology of GI motility and pain disorders. This information would help to develop new drug or other treatments for common motility and visceral pain disorders like the irritable bowel syndrome (IBS).